Simple ELISA Research Articles

Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 μl of standard serum or serum of patients and 200 μl of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37°C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantiti of myoglobin within the range 2–1000 ng/ml serum. 120 samples can be tested in 3 h.

A rapid and simple ELISA for the determination of duplicate monoclonal antibodies during epitope analysis of antigens and its application to the study of [formula omitted

A rapid and simple ELISA has been developed for identifying the specificities of two monoclonal antibodies recognizing either similar or distinct epitope(s) of an antigen. The method utilizes microtiter plates coated with one of the monoclonal antibodies either by direct adsorption of the purified antibody to the plastic or by immobilization of the antibody from ascites or hybridoma supernatants via immobilized polyclonal anti-mouse immunoglobulin antibodies. After preincubation of the antigen with the second monoclonal antibody, the mixture is added to the surface-immobilized first antibody. The amount of antigen bound to the first antibody is subsequently measured by rabbit polyclonal antibodies to the antigen and peroxidase-conjugated anti-rabbit immunoglobulin antibodies. Binding of antigen to the first antibody is only observed when the second monoclonal antibody binds to a distinct epitope. The major advantages of this procedure are its simplicity, rapidity and independence of radioisotopes. Using this method a library of monoclonal antibodies against human C 1- INH has been tested and several duplicate monoclonal antibodies have been identified. Furthermore, the above analytical procedure was capable of detecting conformational changes of the C 1- INH molecule induced either by binding of a monoclonal antibody to C 1- INH or by enzymatic cleavage of C 1- INH .

A sensitive enzyme-linked immunosorbence assay for the c-fos and v-pos oncoproteins

The c-fos nuclear oncoprotein is rapidly induced when the growth of normal cells is initiated by mitogens, and it is also synthesised in several cell systems in response to stimuli that do not cause cell proliferation. When expressed inappropriately, c-fos, and its retroviral counterpart v-pos, can transform susceptible cells in vivo and in vitro. We have developed a simple and sensitive ELISA for the c-fos and v-pos proteins. Fos proteins are captured from cell lysates by an antibody specific for an amino-terminal peptide substantially conserved between v-pos and c-pos; the captured proteins are recognised by a second antibody against a different peptide sequence also conserved in the two proteins. The second antibody has been conjugated to alkaline phosphatase to provide an enzyme label; bound alkaline phosphatase is measured with a sensitive cycling enzyme system that generates a coloured end-product. We show that the fos ELISA is immunologically specific and use it to monitor increased c-fos expression in serum-stimulated HeLa cells and human fibroblasts, and in mitogen-stimulated murine thymocytes.

Development of ELISA for Ophiostoma ulmi using antigen‐coated wells

A simple sensitive ELISA test has been developed that detects either antibodies to, or antigens of, the Dutch elm pathogen, Opiostoma (Ceratocystis) ulmi. The dilution end point for antisera raised in mice againt mycelial antigens was 10‐4. The minimum amount of antigen detected in wells passively coated with antigens solubilized in phosphate‐buffered saline was 500 pg/ml. The assay proved an effective method for rapidly screening large numbers of hybridoma supernatants for monoclonal antibodies that recognize O. ulmi antigens and it has also been used to detect fungal antigens in saline extracts of diseased tissue. Non‐specific binding of preimmune antiserum to plant extracts was markedly higher in extracts from diseased than non‐diseased tissue. Production by the diseased host or by the pathogen in vivo of a protein A or lectin‐like molecule that binds non‐specifically to immunoglobulins is postulated.

A simplified assay for the detection of C3a in human plasma employing a monoclonal antibody raised against denatured C3

A monoclonal antibody raised against SDS-denatured C3 was shown to react with both solid-phase C3a and unfragmented C3. However, in the fluid phase the antibody was found to bind only to C3a and not to native C3. These findings indicated that the antibody could be used in an assay to detect C3a in human EDTA-plasma without prior separation of C3a from native C3. A simple and rapid competition ELISA was developed which monitored soluble C3a. 200 μl of C3a (8 ng) was adsorbed to plastic wells over night at 4°C. Thereafter, 50 μl of sample and 50 μl of constant amounts of monoclonal antibody conjugated with β-galactosidase, were incubated for 60 min at 37°C. After washing, the colour reaction was started by adding nitrophenyl-galactopyridine to the wells. The microtitre plate was incubated at 37°C for 30 min and the staining intensity was quantified at 405 nm. The assay detected both C3a and C3a des arg. A strong correlation was obtained between the new technique and an RIA which used an acid precipitation step for the separation of C3a prior to the determination of C3a ( r = 0.9). Significantly higher levels of C3a were detected both in plasma from patients with immune complexes (93 ± 9 ng/ml; P < 0.1) and in plasma from patients treated in blood oxygenators (140 ± 19 ng/ml; P < 0.05) than in plasma from normal subjects (74 ± 4 ng/ml). The results were not affected by repeated freezing and thawing of the plasma samples.

A simple ELISA for the classification of monoclonal antibodies according to their recognition of native epitopes

A sandwich ELISA for testing whether pairs of monoclonal antibodies recognize the same native antigenic site was developed. The assay was performed with murine anti-lysozyme monoclonal antibodies. A monoclonal antibody, adsorbed onto a microtiter plate, was used as a capture antibody for native lysozyme. After the reaction with the antigen a second monoclonal antibody,the test antibody, was added. The amount of bound antibody was quantitatively measured using rabbit anti-mouse immunoglobulin serum conjugated to alkaline phosphatase. The simultaneous binding of pairs of monoclonal antibodies to lysozyme was further substantiated by structural considerations.

An ELISA for IgA, IgG and IgM-RF measurement. I. Parameters of the assay.

A simple and rapid solid phase ELISA for the stepless determination of IgA, IgG and IgM-RF was developed. The ELISA works with Fc parts of human IgG as antigen. Specificity, validity and precision were tested. RF complex dissociation by urea and separation by gel filtration was performed in several samples to obtain information of the transfer of non-RF-IgG by pentameric IgM-RF. The RF-ELISA may give better information on the significance of RF of the three Ig classes in the clinical course of RA.

Sensitive ELISA for the gp120 and gp160 surface glycoproteins of HIV-1.

We have used a panel of polyclonal and monoclonal antibodies against gp120 and gp160, the envelope glycoproteins of human immunodeficiency virus type 1, to create rapid, simple, and sensitive twin-site sandwich ELISA specific for gp120 and gp160 or for gp160 alone. These assays can detect 500 COS cells in a population transiently transfected with a construct encoding gp120 and gp160, or 50 pg of recombinant gp160. We estimate that the mean amount of gp120 + gp160 in the transfected population is equivalent to 2.5 x 10(6) molecules per cell, 40-50% of which can be recovered from the culture medium as gp120 after 24 hours. The ELISA can be adapted to assess whether gp120 is detectable in the sera of HIV-1-infected persons: we show that gp120/gp160 is completely stable in normal human serum for at least 24 hours at 37 degrees C.

The design of a simple competitive elisa using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene ( pho A ) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5′-terminal end of pho A . In the other construction a 42 base pair DNA fragment has been deleted from the 3′-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10–15 U/mg and 18–25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.

A one-step enzyme-linked immunosorbent assay to detect anti-CMV antibodies; development and clinical validation

A simple one-step ELISA to detect anti-CMV antibodies was developed. The test was based on the inhibition principle, and used anti-CMV coated microtitre plates, CMV nuclear antigen and anti-CMV Fab'-HRP conjugate; total assay time was 1.5 h. The use of Fab'-HRP conjugates improved discrimination between positive and negative sera as compared with IgG-HRP conjugates. In an in-house clinical study, the one-step ELISA showed a 98.4% correlation with the latex agglutination test. The test could be demonstrated to be suitable for both serum and citrate- or EDTA-plasma specimens; heparin-plasma gave somewhat higher absorbance values. In an external clinical validation, the one-step ELISA showed a 99.5% correlation with the latex agglutination test, and was found to be highly suitable for screening of blood donor specimens in a blood bank setting.

Studies on HBsAg binding with polymerised human serum albumin by ELISA

A simple and sensitive ELISA was developed to characterize the interaction between polymerised human serum albumin (pHSA) and HBsAg, using pHSA-coated polyvinylmicrotitre plates as solid phase and anti-HBs-coupled HRPO as the conjugate. The interaction was found to be specific and dependent on the size of albumin polymer. pHSA-binding activity (pHSA-BA) was studied in both HBsAgnegative and HBsAg-positive sera from various liver diseases including acute viral hepatitis, fulminant hepatitis, cirrhosis of liver, chronic active hepatitis, and healthy HBsAg carriers. pHSA-BA was detected only in HBsAg-positive sera. Analysis of HBsAg-positive sera indicated pHSA-BA in high proportions of patients sera as compared to sera from healthy HBsAg carriers. pHSA-BA was detected both in the presence and absence of HBe markers, though the mean BA was relatively high in presence of HBeAg. The effect of human serum immunoglobulins (IgG, IgA, and IgM) on the BA was investigated and a correlation between pHSA-BA and HBsAg-IgM complex positivity in sera was established. Finally, the probable role of human serum IgM in facilitating the binding process was discussed.

A low pH enzyme linked immunoassay using two monoclonal antibodies for the serological detection and monitoring of breast cancer.

A new, simple and sensitive low pH ELISA method has been developed to measure serum levels of tumour associated antigens detectable by monoclonal antibodies HMFG1 and HMFG2. We examined sera from healthy controls, patients with neoplastic and non-neoplastic conditions of breast, liver and gastrointestinal tract. The majority of patients with metastatic breast cancer had elevated serum antigens (69% HMFG1, 72% HMFG2) compared to healthy controls (6.3% HMFG1, 3.0% HMFG2) or patients with benign breast disease (17% HMFG1, 4% HMFG2). There was no discrimination using these assays between patients with neoplastic and non-neoplastic conditions of liver and gastrointestinal tract. This new method promises to be of value in the assessment and management of patients with breast cancer.

A rapid ELISA for measurement of antibodies to nucliec acid antigens using UV-treated polystyrene microplates

Pretreatment of polystyrene microplate wells with certain doses of UV light enhances their capacity for binding to single-stranded DNA, double stranded DNA and various synthetic polynucleotides. The use of UV-irradiated plates to immobilize nucleic acid antigens provides a simple, rapid, and specific ELISA for measuring anti-nucleic acid antibodies. The assay is at least as sensitive as the more complex method of precoating plates with poly( L-lysine). It is useful for detection of anti-DNA antibodies in sera of systemic lupus erythematosus patients, as well as in culture fluids of murine and human anti-DNA-secreting hybridomas.

A simple and sensitive ELISA to detect immune (γ) interferon induced I-A on a macrophage line

A convenient and sensitive cell surface ELISA is described to detect immune (γ) interferon induced I-A determinants on a murine macrophage line, P388D 1. The ELISA is performed on monolayers of P388D 1 cells grown in 96-well culture plates for 2 days in the presence of recombinant γIFN or supernatants from cultures of antigen-activated T cells. After glutaraldehyde fixation, the cultured cells are overlayered with fetal calf serum to block nonspecific antibody binding during the assay. A double sandwich technique employing a monoclonal anti-I-A d (MKD6) antibody followed by peroxidase-conjugated goat anti-mouse γ chain antibody is then used to detect the presence of I-A on the cell monolayers. Detection of I-A induced by both rγIFN and the supernatant from an antigen-stimulated T cell line was highly reproducible and sensitive using this method. Furthermore, the assay is easily performed and many sample supernatants can be rapidly screened for this activity. The assay is used by this laboratory to detect the antigen-stimulated production of γIFN by T cell clones and hybridomas.

Rapid Serological Diagnosis of Mumops Virus Infection by an Enzyme-Linked Immunosorbent Assay of Mumps IgG and IgM Antibodies

A simple and sensitive ELISA of mumps IgM and IgG antibodies was described. This assay served as a useful means of rapid diagnosis of mumps virus infection. Coolection of parired serum specimens before day 2 and during the second week of the disease was recommended. A great increase in mumps ELISA IgG in combination with the presence of significant elevation of mumps ELISA IgM level was one of the best proffs of quite recent mumps virus infection. Infulences of false negative reaations and false positive reactions on mumps ELISA IgM were not great. We concluded that the measurement of ELISA IgM antibody along with that of IgG antibody was useful for rapid serological diagnosis of recent mumps infection.

An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility. specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.

A monoclonal antibody directed against an organ-specific liver cell membrane antigen in rabbits

We generated monoclonal antibodies after immunization of mice with rabbit liver-specific protein (LSP) preparations. One of these antibodies (2D3) showed an organ-specific and species-specific binding pattern as determined by immunohistological and ELISA techniques. Immunoelectromicroscopy studies demonstrated that this antibody is bound exclusively to the liver cell membrane except in the region of the bile canaliculi. We further describe a simple ELISA technique for the detection of anti-LSP antibodies. Our study clearly demonstrates the presence of at least 1 organ-specific liver cell membrane antigen in rabbit LSP and shows antigenic differences between different areas of the plasma membrane of hepatocytes.